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Identification of novel high-frequency DNA methylation changes in breast cancer.

Ordway JM, Budiman MA, Korshunova Y, Maloney RK, Bedell JA, Citek RW, Bacher B, Peterson S, Rohlfing T, Hall J, Brown R, Lakey N, Doerge RW, Martienssen RA, Leon J, McPherson JD, Jeddeloh JA - PLoS ONE (2007)

Bottom Line: Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis.The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively.Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date.

View Article: PubMed Central - PubMed

Affiliation: Orion Genomics, St. Louis, Missouri, United States of America. jordway@oriongenomics.com

ABSTRACT
Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

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Related in: MedlinePlus

Differential DNA methylation of identified loci within an initial validation panel of clinical samples.qPCR measurements of DNA methylation density were obtained for 53 loci across 16 IDC tumor samples and 25 normal or benign breast samples. An average delta Ct (Ct McrBC–Ct Mock) less than 1.0 was scored as sparsely methylated (green cells). A delta Ct of 1.0 indicates that approximately half of the DNA in the reaction was cleaved by McrBC within the amplified region and therefore contained a measurable density of DNA methylation. An average delta Ct greater than or equal to 1.0, but less than 2.0 was scored as intermediately methylated (yellow cells). Finally, an average delta Ct≥2.0 (≥75% of DNA molecules were cleaved by McrBC) was scored as densely methylated (red cells).
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pone-0001314-g003: Differential DNA methylation of identified loci within an initial validation panel of clinical samples.qPCR measurements of DNA methylation density were obtained for 53 loci across 16 IDC tumor samples and 25 normal or benign breast samples. An average delta Ct (Ct McrBC–Ct Mock) less than 1.0 was scored as sparsely methylated (green cells). A delta Ct of 1.0 indicates that approximately half of the DNA in the reaction was cleaved by McrBC within the amplified region and therefore contained a measurable density of DNA methylation. An average delta Ct greater than or equal to 1.0, but less than 2.0 was scored as intermediately methylated (yellow cells). Finally, an average delta Ct≥2.0 (≥75% of DNA molecules were cleaved by McrBC) was scored as densely methylated (red cells).

Mentions: As an initial validation, qPCR assays for 53 loci hypermethylated in at least 70% of tissue pairs were conducted across a panel of 16 independent IDCs (Stage II) and 25 normal or benign breast tissues. We focused on hypermethylation events because hypomethylation events were considerably less frequent than hypermethylation events (i.e. <40% sensitivity). This observation has been made previously by Bestor and colleagues (A. O'Donnell, R. Rollins, and T.H. Bestor (personal communication)). As shown in Fig. 3, differential DNA methylation between tumor and non-tumor breast samples was confirmed in an independent tissue panel. The differentially methylated regions displayed a range of clinical sensitivity (i.e. the percentage of tumors displaying intermediate to dense DNA methylation) and clinical specificity (i.e. the percentage of normals displaying sparse DNA methylation). Among loci displaying 100% specificity relative to normal breast tissue, sensitivities ranged from 6% (1 of 16 tumors were methylated; IGF-II mRNA binding protein 3) to 81% (13 of 16 tumors were methylated; GHSR) (Table S3). Because these differentially methylated loci may be useful for disease detection in peripheral fluids such as plasma or serum, the methylation status of each locus was analyzed in a panel of 19 blood samples from cancer-free women. Although the majority of the 53 loci demonstrated greater than 80% specificity relative to normal peripheral blood, 21 loci (40%) were methylated in at least half of the normal blood samples (Table S3). Therefore, these results indicate that a subset of loci that become hypermethylated in breast cancer take on a DNA methylation state that is similar to the normal methylation state in circulating blood cells. Similar results were obtained in a recent DNA methylation analysis of lung tumors and peripheral blood [28]. Although the biological mechanisms and consequences of the DNA methylation similarities between tumor and normal peripheral blood cells are yet to be determined, these findings are important in terms of the applicability of differentially methylated loci for use as potential biomarkers for early detection of cancer using peripheral fluids such as serum or plasma


Identification of novel high-frequency DNA methylation changes in breast cancer.

Ordway JM, Budiman MA, Korshunova Y, Maloney RK, Bedell JA, Citek RW, Bacher B, Peterson S, Rohlfing T, Hall J, Brown R, Lakey N, Doerge RW, Martienssen RA, Leon J, McPherson JD, Jeddeloh JA - PLoS ONE (2007)

Differential DNA methylation of identified loci within an initial validation panel of clinical samples.qPCR measurements of DNA methylation density were obtained for 53 loci across 16 IDC tumor samples and 25 normal or benign breast samples. An average delta Ct (Ct McrBC–Ct Mock) less than 1.0 was scored as sparsely methylated (green cells). A delta Ct of 1.0 indicates that approximately half of the DNA in the reaction was cleaved by McrBC within the amplified region and therefore contained a measurable density of DNA methylation. An average delta Ct greater than or equal to 1.0, but less than 2.0 was scored as intermediately methylated (yellow cells). Finally, an average delta Ct≥2.0 (≥75% of DNA molecules were cleaved by McrBC) was scored as densely methylated (red cells).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2117343&req=5

pone-0001314-g003: Differential DNA methylation of identified loci within an initial validation panel of clinical samples.qPCR measurements of DNA methylation density were obtained for 53 loci across 16 IDC tumor samples and 25 normal or benign breast samples. An average delta Ct (Ct McrBC–Ct Mock) less than 1.0 was scored as sparsely methylated (green cells). A delta Ct of 1.0 indicates that approximately half of the DNA in the reaction was cleaved by McrBC within the amplified region and therefore contained a measurable density of DNA methylation. An average delta Ct greater than or equal to 1.0, but less than 2.0 was scored as intermediately methylated (yellow cells). Finally, an average delta Ct≥2.0 (≥75% of DNA molecules were cleaved by McrBC) was scored as densely methylated (red cells).
Mentions: As an initial validation, qPCR assays for 53 loci hypermethylated in at least 70% of tissue pairs were conducted across a panel of 16 independent IDCs (Stage II) and 25 normal or benign breast tissues. We focused on hypermethylation events because hypomethylation events were considerably less frequent than hypermethylation events (i.e. <40% sensitivity). This observation has been made previously by Bestor and colleagues (A. O'Donnell, R. Rollins, and T.H. Bestor (personal communication)). As shown in Fig. 3, differential DNA methylation between tumor and non-tumor breast samples was confirmed in an independent tissue panel. The differentially methylated regions displayed a range of clinical sensitivity (i.e. the percentage of tumors displaying intermediate to dense DNA methylation) and clinical specificity (i.e. the percentage of normals displaying sparse DNA methylation). Among loci displaying 100% specificity relative to normal breast tissue, sensitivities ranged from 6% (1 of 16 tumors were methylated; IGF-II mRNA binding protein 3) to 81% (13 of 16 tumors were methylated; GHSR) (Table S3). Because these differentially methylated loci may be useful for disease detection in peripheral fluids such as plasma or serum, the methylation status of each locus was analyzed in a panel of 19 blood samples from cancer-free women. Although the majority of the 53 loci demonstrated greater than 80% specificity relative to normal peripheral blood, 21 loci (40%) were methylated in at least half of the normal blood samples (Table S3). Therefore, these results indicate that a subset of loci that become hypermethylated in breast cancer take on a DNA methylation state that is similar to the normal methylation state in circulating blood cells. Similar results were obtained in a recent DNA methylation analysis of lung tumors and peripheral blood [28]. Although the biological mechanisms and consequences of the DNA methylation similarities between tumor and normal peripheral blood cells are yet to be determined, these findings are important in terms of the applicability of differentially methylated loci for use as potential biomarkers for early detection of cancer using peripheral fluids such as serum or plasma

Bottom Line: Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis.The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively.Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date.

View Article: PubMed Central - PubMed

Affiliation: Orion Genomics, St. Louis, Missouri, United States of America. jordway@oriongenomics.com

ABSTRACT
Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

Show MeSH
Related in: MedlinePlus